The Sinorhizobium meliloti EmrR Regulator Is Required for Efficient Colonization of Medicago sativa Root Nodules

The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse conditions encountered during its symbiosis with leguminous plants. We characterized a new symbiotically relevant gene, emrR (SMc03169), whose product belongs to the TetR family of repressors and is divergently transcribed from emrAB genes encoding a putative major facilitator superfamily-type efflux pump. An emrR deletion mutant produced more succinoglycan, displayed increased cell-wall permeability, and exhibited higher tolerance to heat shock. It also showed lower tolerance to acidic conditions, a reduced production of siderophores, and lower motility and biofilm formation. The simultaneous deletion of emrA and emrR genes restored the mentioned traits to the wild-type phenotype, except for survival under heat shock, which was lower than that displayed by the wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules and competed poorly with the wild-type strain for nodule colonization. Expression profiling of the ΔemrR mutant showed decreased expression of genes involved in Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of genes directing the biosynthesis of succinoglycan and other polysaccharides were increased. EmrR may therefore be involved in a regulatory network targeting membrane and cell wall modifications in preparation for colonization of root hairs during symbiosis.

Ecology of west Iberian kelps: Laminaria ochroleuca and Saccorhiza polyschides living near their distributional limits

Tese de Doutoramento, Ecologia (Ecologia das Populações), Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Loss of thermal refugia near equatorial range limits

This study examines the importance of thermal refugia along the majority of the geographical range of a key inter- tidal species (Patella vulgata Linnaeus, 1758) on the Atlantic coast of Europe. We asked whether differences between sun-exposed and shaded microhabitats were responsible for differences in physiological stress and ecological perfor- mance and examined the availability of refugia near equatorial range limits. Thermal differences between sun- exposed and shaded microhabitats are consistently associated with differences in physiological performance, and the frequency of occurrence of high temperatures is most probably limiting the maximum population densities sup- ported at any given place. Topographical complexity provides thermal refugia throughout most of the distribution range, although towards the equatorial edges the magnitude of the amelioration provided by shaded microhabitats is largely reduced. Importantly, the limiting effects of temperature, rather than being related to latitude, seem to be tightly associated with microsite variability, which therefore is likely to have profound effects on the way local popu- lations (and consequently species) respond to climatic changes.

Caracterisation fonctionelle d'un facteur d'elongation mitochondrial leEF-Tsmt chez la tomate: approaches par transgenese et proteomique.

Esta tese é relacionada ao estudo funcional de um gene que codifica um fator de elongação LeEF-Tsmt em tomate. Este gene participa no processo de síntese de proteína em mitocôndrias e apresenta uma forte expressão durante o processo de maturação quando comparado a outros órgãos. Nós demonstramos que o mesmo se exprime fortemente durante as primeiras fases do processo maturação em paralelo com a crise respiratória climatérica e que sua expressão é estimulada pelo etileno, ferimento e altas temperaturas. Porém, os mutantes de tomate insensíveis ao etileno, exibem uma expressão normal. Frutos transgênicos foram gerados, nos quais o LeEF-Tsmt foi aumentado ou inibido de uma forma constitutiva. Porém, a alteração da expressão do gene através da transformação genética com construções sentido e antisense do gene LeEF-Tsmt não afeta o padrão de respiração e produção de etileno durante a maturação e após o ferimento. Além disso, a expressão do gene da alternativa oxidase, que é conhecida por apresentar um papel importante no climatério respiratório, não foi afetada. Todos estes dados indicam que apesar de sua forte regulação, o LeEF-Tsmt não é limitante da atividade respiratória mitocondrial. A expressão do gene de LeEF-Tsmt é estimulada pelo efeito do estresse oxidativo induzido nas partes vegetativas da planta pela seca e o paraquat. A sensibilidade ao estresse oxidativo avaliado em folhas pela presença de necrose e em calos através de crescimento celular, foi reduzido em plantas antisentido. Entre as enzimas conhecidas por apresentar um papel na detoxificação de espécies reativas de oxigênio, superóxido dismutase (SOD), catalases (CAT), peroxidase (PX) e glutation redutase (GR), nós demostramos que a GR e PX exibem atividade mais alta em linhas antisentido, explicando assim, pelo menos em parte, sua melhor tolerância ao estresse. O papel da proteína de LeEF-Tsmt na síntese de proteínas mitocondriais foi estudado pela análise do proteôma mitocondrial em linhas antisentido e sentido do gene LeEF-Tsmt. A comparação dos proteômas de linhas transformadas e selvagem foi tratado com a ajuda de uma técnica de dupla marcagem 14N/15N aplicadas à tecidos de tomate cultivados in vitro. A linha sentido super expressa fortemente a proteína, enquanto que as linhas antisentidos diminuem ligeiramente. Uma proteína do tipo ?heat-shock? segue as variações da proteína LeEF-Tsmt, sugerindo um possível papel chaperona. Uma análise global do proteôma mitocondrial foi executada, fornecendo novas informações sobre um conjunto de ao redor 500 proteínas mitocondriais de tomate.

Changes in enzymatic activity, accumulation of proteins and softening of persimmon (Diospyros kaki Thunb.) flesh as a function of pre-cooling acclimatization.

One of the major causes of ?Fuyu? persimmon loss after cold storage (CS) is the breakdown of its flesh, which results in the production of a translucent fruit (a water-soaked fruit). It is believed that the cause of this disturbance is linked to disorganization of the cytoskelet and endomembrane system, which changes the synthesis and transport of proteins and metabolites, resulting in incomplete ripening. To test this hypothesis, ?Fuyu? persimmon was subjected to three different postharvest treatments (T): Control ? harvested and kept at 23±3 702;C and relative humidity (RH) of 85±5% (room temperature, RT) for 12 days, T1 ? harvested and kept under cold storage (CS) (1±1 702;C and RH of 85±5%) for 30 days followed by RT storage for 2 days, T2 ? kept under RT for 2 days (acclimatization) followed by CS for 30 days. Control and T2 resulted in fruit with decreased flesh firmness (FF), and increased soluble solids (SS) and ascorbic acid (AA) contents. In these fruit the activity of endo-1,4-ß-glucanase (endo-1,4-ß-gluc), pectin methylesterase (PME), polygalacturonase (PG) and ß-galactosidase (ß-gal) increased. T1 resulted in translucent fruit with decreased FF, without any enzymatic activity changes, probably due to the physical disruption of the cytoskeleton. Further, there was an increased content of proteins corresponding to expansins in fruit kept under Control and T2 conditions, which suggests that these conditions do contribute to the synthesis and/or transport of proteins involved in the process of solubilization of the cell wall. In these fruit, there was also a major accumulation of gene transcripts corresponding to heat shock proteins (HSPs) of organelles related to endomembrane, which suggests participation of these genes in the prevention of damage caused by cold conditions. These data proved the hypotheses that acclimatization contributes to the expression of HSPs, and synthesis and transportat of proteins involved in the solubilization of the cell wall. The expression of these genes results in the normal ripening of the persimmon, as confirmed by the evolution of ethylene production.

Cutaneous vasculitis in systemic lupus erythematosus: association with anti-ribosomal P protein antibody and Raynaud phenomenon

Ninety-one consecutive systemic lupus erythematosus (SLE) patients (American College of Rheumatology criteria) with a history of cutaneous vasculitis were compared to 163 SLE controls without this clinical manifestation from July to December 2007 in order to determine the possible clinical and serological association of this manifestation. Data were obtained in an ongoing electronic database protocol and autoantibodies to anti-double-stranded DNA, anti-Sm, anti-RNP, anti-Ro/SS-A, anti-La/SS-B, and anticardiolipin and ribosomal P protein antibody (anti-P) were detected by standard techniques. Exclusion criteria were the presence of anti-phospholipid syndrome or antibodies, Sjogren syndrome, and a history of thrombosis. The mean age (38.5 +/- 11.5 vs. 37.8 +/- 11.6 years, p = 0.635), disease duration (12.5 +/- 7.8 vs. 11.8 +/- 7.9 years, p = 0.501), and frequency of white race (71.4% vs. 70.5%, p = 0.872) and female sex (96.8% vs. 93.7%, p = 0.272) were comparable in both groups. The vasculitis group had a higher frequency of malar rash (97.9% vs. 87.4%, p = 0.004), photosensitivity (91.4% vs. 81.6%, p = 0.030), and Raynaud phenomenon (RP; 27.7% vs. 7.5%, p < 0.001), whereas all other clinical manifestation including renal and central nervous system involvements were similar to the control group. Laboratorial data revealed that only anti-P (35.1% vs. 12.1%, p < 0.001) was more frequent in patients with vasculitis. In a multivariate logistic regression model, cutaneous vasculitis was associated to the presence of RP (OR = 3.70; 95% confidence interval [CI] = 1.73-8.00) and anti-P (OR = 3.42; 95% CI = 1.76-6.66). In summary, SLE cutaneous vasculitis characterizes a subgroup of patients with more RP and anti-P antibodies but not accompanied by a higher frequency of renal and central nervous system involvements.

Antibody response to heat-inactivated hepatitis B vaccine (CLB-3mg) in hemodialysis patients and occupational risk personnel: a one year follow-up

Immune response against hepatitis B vaccine (CLB 3mg) was evaluated in 59 hemodialysis patients and 20 occupational risk personnel. Seroconversion was induced in 52.5% and 70.0% respectively. Twelve months after the first dose, 37.5% of patients and 60.0% of occupational risk personnel had detectable anti-HBs level. Antibody level was expressed in sample ratio units (SRU). Considering only the responders, in the patients group 38.7% had a low anti-HBs response (2.1-9.9 SRU) 32.3% a medium response (10-99.9 SRU) and 29.0% a high response (>100 SRU) while in occupational risk personnel these values were 14.3%, 64.3% and 21.4% respectively. The authors suggest the use of HBV vaccines with more elevated HBsAg concentration or a reinforced immunization schedule to improve the anti-HBs response not only for patients but also for healthy persons.

Activated protein C consumption and coagulation parameters in severe sepsis and septic shock.

Introduction Activated protein C (APC) deC ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24< hours from severe sepsis or septic shock onset, deC ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.370.7% had medical pathology. The 28-day mortality was 22.7%. Nonsurvivors had a signiC cantly higher consumption of APC than survivors, 56% (IQR: 38.5) versus 68.6 (IQR: 41.4); P = 0.023. The proC le of lower levels of APC was a surgery patient with septic shock, neurological focus or catheter-related infection and Gram-negative pathogens from blood cultures. Spearman’s showed relationship with antithrombin III, r<= 0.674 (P <0.001) and International Normalized Ratio (INR), r<= –0.611 (P >0.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR.

A phase IIa randomized clinical study testing GNbAC1, a humanized monoclonal antibody against the envelope protein of multiple sclerosis associated endogenous retrovirus in multiple sclerosis patients - A twelve month follow-up.

GNbAC1 is a humanized monoclonal antibody targeting MSRV-Env, an endogenous retroviral protein, which is expressed in multiple sclerosis (MS) lesions, is pro-inflammatory and inhibits oligodendrocyte precursor cell differentiation. This paper describes the open-label extension up to 12months of a trial testing GNbAC1 in 10 MS patients at 2 and 6mg/kg. The primary objective was to assess GNbAC1 safety, and other objectives were pharmacokinetic and pharmacodynamic assessments. During the extended study, no safety issues occurred in the 8 remaining patients. No anti-GNbAC1 antibodies were detected. GNbAC1 appears well tolerated.

Cold-induced enhancement of avian uncoupling protein expression, heat production, and triiodothyronine concentrations in broiler chicks

The relationships among avian uncoupling protein (avUCP) mRNA expression, heat production, and thyroid hormone metabolism were investigated in 7-14-day-old broiler chicks (Gallus gallus) exposed to a low temperature (cold-exposed chicks, CE) or a thermoneutral temperature (TN). After 7 days of exposure, CE chicks exhibited higher heat production (+83%, P < 0.01), avUCP mRNA expression (+20%, P < 0.01), and circulating triiodothyronine (T-3) levels (+104%, P = 0.07) for non-statistically different body weights and feed intake between 3 and 7 days of exposure as compared to TN chicks. Plasma thyroxine (T-4) concentration was clearly decreased in CE chicks (-33%, P = 0.06). The lower hepatic inner-ring deiodination activity (-47%) and the higher renal outer-ring deiodination activity (+75%) measured in CE compared to TN chicks could partly account for their higher plasma T3 concentrations. This study describes for the first time the induction of avUCP mRNA expression by low temperature in chickens, as it has been previously shown in ducklings, and supports the possible involvement of avUCP in avian thermogenesis. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Net energy, protein and macrominerals requirements for 70 to 120 day old female rabbits

In order to determine the net energy, protein and macrominerals requirements of 70 to 120 day old, 52 female White New Zealand rabbits, weighing 1900g +/- 40g were used. At the beginning of the experimental period, 14 of the 52 young does were slaughtered and the 38 remaining animals were kept under two dietary management: ad libitum and restricted feeding. Slaughters were performed to determine each nutrient body content. The weight gain nutrient requirements depicted by the quantities of each nutrient stored into the body were obtained by applying the regression equation, which estimate the empty body nutrient content logarithm as a function of the empty body weight logarithm, as described by ARC (1980). By determining the heat production logarithm at the zero level of metabolizable energy intake, the maintenance net energy requirement was estimated to be 45.31 Kcal/day/Kg(0.75) the mean net energy. protein, calcium, phosphorous, sodium, magnesium and potassium requirements for each gram of weight gain per day were estimated to be, 2.51 Kcal, 0.21g, 0.02g, 0.005g, 0.001g, 0.0004g and 0.002g, respectively.

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LTG33D), originally produced by some enterotoxigenic Escherichia coil (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LIG33D. Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LTG33D elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LTG33D. Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LTG33D. Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine. (C) 2011 Elsevier Ltd. All rights reserved.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon’s amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

Comparison of C-reactive protein and serum amyloid A protein in septic shock patients

Septic shock is a severe inflammatory state caused by an infectious agent. Our purpose was to investigate serum amyloid A (SAA) protein and C-reactive protein (CRP) as inflammatory markers of septic shock patients. Here we evaluate 29 patients in postoperative period, with septic shock, in a prospective study developed in a surgical intensive care unit. All eligible patients were monitored over a 7-day period by sequential organ failure assessment (SOFA) score, daily CRP, SAA, and lactate measurements. CRP and SAA strongly correlated up to the fifth day of observation but were not good predictors of mortality in septic shock. Copyright (C) 2008.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

Background: Celery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model. Methodology: 786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs. Results: IgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides. Conclusions: Api g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.